Transition from Colitis to Cancer: High Wnt Activity Sustains the Tumor-Initiating Potential of Colon Cancer Stem Cell Precursors

Abstract

Ulcerative colitis (UC) increases the risk of colorectal cancer (CRC), but the mechanisms involved in colitis-to-cancer transition (CCT) are not well understood. CCT may involve a inflammation-dysplasia-carcinoma progression sequence compared to the better characterized adenoma-carcinoma progression sequence associated with sporadic CRC. One common thread may be activating mutations in components of the Wnt/β-catenin signaling pathway, which occur commonly as early events in sporadic CRC. To examine this hypothesis, we evaluated possible associations between Wnt/β-catenin signaling and CCT based on the cancer stem cell (CSC) model. Wnt/β-catenin immunostaining indicated that UC patients have a level of Wnt-pathway-active cells that is intermediate between normal colon and CRC. These UC cells exhibiting activation of the Wnt pathway constituted a major subpopulation (52%+7.21) of the colonic epithelial cells positive for aldehyde dehydrogenase (ALDH), a putative marker of precursor colon CSC (pCCSC). We further fractionated this subpopulation of pCCSC using a Wnt pathway reporter assay. Over successive passages, pCCSCs with the highest Wnt activity exhibited higher clonogenic and tumorigenic potential than pCCSCs with the lowest Wnt activity, thereby establishing the key role of Wnt activity in driving CSC-like properties in these cells. Notably, 5/20 single cell injections of high-Wnt pCCSC resulted in tumor formation, suggesting a correlation with CCT. Attenuation of Wnt/β-catenin in high-Wnt pCCSC by shRNA-mediated downregulation or pharmacological inhibition significantly reduced tumor growth rates. Overall, the results of our study indicates (i) that early activation of Wnt/β-catenin signaling is critical for CCT, and (ii) that high levels of Wnt/β-catenin signaling can further demarcate ALDH+ tumor-initiating cells in the non-dysplastic epithelium of UC patients. As such, our findings offer plausible diagnostic markers and therapeutic target in the Wnt signaling pathway for early intervention in CCT.

Morphological Classification of Corrupted Colonic Crypts in Ulcerative Colitis

“…In closing, while cellular descriptions and molecular signals that control cell proliferation and cell mutations during carcinogenesis in UC have received much attention ( [35][36][37][38][39][40], the signals that might be instrumental in designing morphogenesis (41) resulting in the "etching" of various CCC phenotypes in UC, have remained unattended.…”

Drug-eluting microarrays to identify effective chemotherapeutic combinations targeting patient-derived cancer stem cells

“…Colon cancer stem cells were generated by isolating and enriching for highlevel aldehyde dehydrogenase (ALDH high ) spheres from colon cancer tumor cells obtained from patients with sporadic colorectal cancer (26,27,29,42). Isolated cells were cultured in serum-free media as described previously (26).…”

“…Isolated cells were cultured in serum-free media as described previously (26). Heterogeneity (as defined by the ability to recapitulate all cellular aspects of a tumor) in CCSCs was maintained (27,29). The isolates maintained in culture originated directly from patients, were propagated and expanded as patientderived xenografts in mice, and were maintained in vitro.…”

“…Recent identification of unique cell surface markers that enrich tumor cell isolates for CSCs have led to novel techniques for isolating enriched colorectal CSC (CCSC) populations from patient tumor samples (25)(26)(27)(28). For example, xenotransplantation of a single CCSC identified by high Wnt/Β-catenin signaling activity generates tumors that recapitulate the diverse phenotypic heterogeneity of the original tumor (29). Thus, identifying and isolating CCSCs out of the tumor bulk from an individual cancer patient and determining sensitivity to chemotherapeutic drugs in vitro is possible (30,31).…”

“…The ALDH high cells have the definitive CSC property of self-renewal, as demonstrated by the gold standard assay of limiting dilution tumor xenograft analysis (26,27,29,(40)(41)(42)(43). In addition, tumor heterogeneity is maintained, as defined by the ability to recapitulate all cellular aspects of the primary tumor (27,29). The CCSC isolates can be serially propagated as tumor xenografts or as nonadherent spheres in vitro.…”